Subgenome evolution in allotetraploid plants.

Polyploidization is a well-known speciation and adaptation mechanism. Traces of former polyploidization events were discovered within many genomes, and especially in plants. Allopolyploidization by interspecific hybridization between two species is common. Among hybrid plants, many are domesticated species of agricultural interest and many of their genomes and of their presumptive parents have been sequenced. Hybrid genomes remain challenging to analyse because of the presence of multiple subgenomes. The genomes of hybrids often undergo rearrangement and degradation over time. Based on 10 hybrid plant genomes from six different genera, with hybridization dating from 10,000 to 5 million years ago, we assessed subgenome degradation, subgenomic intermixing and biased subgenome fractionation. The restructuring of hybrid genomes does not proceed proportionally with the age of the hybrid. The oldest hybrids in our data set display completely different fates: whereas the subgenomes of the tobacco plant Nicotiana benthamiana are in an advanced stage of degradation, the subgenomes of quinoa (Chenopodium quinoa) are exceptionally well conserved by structure and sequence. We observed statistically significant biased subgenome fractionation in seven out of 10 hybrids, which had different ages and subgenomic intermixing levels. Hence, we conclude that no correlation exists between biased fractionation and subgenome intermixing. Lastly, domestication may encourage or hinder subgenome intermixing, depending on the evolutionary context. In summary, comparative analysis of hybrid genomes and their presumptive parents allowed us to determine commonalities and differences between their evolutionary fates. In order to facilitate the future analysis of further hybrid genomes, we automated the analysis steps within manticore, which is publicly available at https://github.com/MatteoSchiavinato/manticore.git.

Parental origin of the allotetraploid tobacco Nicotiana benthamiana.

Nicotiana section Suaveolentes is an almost all-Australian clade of allopolyploid tobacco species including the important plant model Nicotiana benthamiana. The homology relationships of this clade and its formation are not completely understood. To address this gap, we assessed phylogenies of all individual genes of N. benthamiana and the well studied N. tabacum (section Nicotiana) and their homologues in six diploid Nicotiana species. We generated sets of 44 424 and 65 457 phylogenetic trees of N. benthamiana and N. tabacum genes, respectively, each collectively called a phylome. Members of Nicotiana sections Noctiflorae and Sylvestres were represented as the species closest to N. benthamiana in most of the gene trees. Analyzing the gene trees of the phylome we: (i) dated the hybridization event giving rise to N. benthamiana to 4-5 MyA, and (ii) separated the subgenomes. We assigned 1.42 Gbp of the genome sequence to section Noctiflorae and 0.97 Gbp to section Sylvestres based on phylome analysis. In contrast, read mapping of the donor species did not succeed in separating the subgenomes of N. benthamiana. We show that the maternal progenitor of N. benthamiana was a member of section Noctiflorae, and confirm a member of section Sylvestres as paternal subgenome donor. We also demonstrate that the advanced stage of long-term genome diploidization in N. benthamiana is reflected in its subgenome organization. Taken together, our results underscore the usefulness of phylome analysis for subgenome characterization in hybrid species.

Genome and transcriptome characterization of the glycoengineered Nicotiana benthamiana line ΔXT/FT.

BACKGROUND: The allotetraploid tobacco species Nicotiana benthamiana native to Australia has become a popular host for recombinant protein production. Although its usage grows every year, little is known on this plant's genomic and transcriptomic features. Most N. benthamiana accessions currently used in research lack proper documentation of their breeding history and provenance. One of these, the glycoengineered N. benthamiana line ΔXT/FT is increasingly used for the production of biopharmaceutical proteins. RESULTS: Based on an existing draft assembly of the N. benthamiana genome we predict 50,516 protein -encoding genes (62,216 transcripts) supported by expression data derived from 2.35 billion mRNA-seq reads. Using single-copy core genes we show high completeness of the predicted gene set. We functionally annotate more than two thirds of the gene set through sequence homology to genes from other Nicotiana species. We demonstrate that the expression profiles from leaf tissue of ΔXT/FT and its wild type progenitor only show minimal differences. We identify the transgene insertion sites in ΔXT/FT and show that one of the transgenes was inserted inside another predicted gene that most likely lost its function upon insertion. Based on publicly available mRNA-seq data, we confirm that the N. benthamiana accessions used by different research institutions most likely derive from a single source. CONCLUSIONS: This work provides gene annotation of the N. benthamiana genome, a genomic and transcriptomic characterization of a transgenic N. benthamiana line in comparison to its wild-type progenitor, and sheds light onto the relatedness of N. benthamiana accessions that are used in laboratories around the world.

Haplotype selection as an adaptive mechanism in the protozoan pathogen Leishmania donovani.

The parasite Leishmania  donovani causes a fatal disease termed visceral leishmaniasis. The process through which the parasite adapts to environmental change remains largely unknown. Here we show that aneuploidy is integral for parasite adaptation and that karyotypic fluctuations allow for selection of beneficial haplotypes, which impact transcriptomic output and correlate with phenotypic variations in proliferation and infectivity. To avoid loss of diversity following karyotype and haplotype selection, L. donovani utilizes two mechanisms: polyclonal selection of beneficial haplotypes to create coexisting subpopulations that preserve the original diversity, and generation of new diversity as aneuploidy-prone chromosomes tolerate higher mutation rates. Our results reveal high aneuploidy turnover and haplotype selection as a unique evolutionary adaptation mechanism that L. donovani uses to preserve genetic diversity under strong selection. This unexplored process may function in other human diseases, including fungal infection and cancer, and stimulate innovative treatment options.

Oligoadenylation of 3' decay intermediates promotes cytoplasmic mRNA degradation in Drosophila cells.

Post-transcriptional 3' end addition of nucleotides is important in a variety of RNA decay pathways. We have examined the 3' end addition of nucleotides during the decay of the Hsp70 mRNA and a corresponding reporter RNA in Drosophila S2 cells by conventional sequencing of cDNAs obtained after mRNA circularization and by deep sequencing of dedicated libraries enriched for 3' decay intermediates along the length of the mRNA. Approximately 5%-10% of 3' decay intermediates carried nonencoded oligo(A) tails with a mean length of 2-3 nucleotides. RNAi experiments showed that the oligoadenylated RNA fragments were intermediates of exosomal decay and the noncanonical poly(A) polymerase Trf4-1 was mainly responsible for A addition. A hot spot of A addition corresponded to an intermediate of 3' decay that accumulated upon inhibition of decapping, and knockdown of Trf4-1 increased the abundance of this intermediate, suggesting that oligoadenylation facilitates 3' decay. Oligoadenylated 3' decay intermediates were found in the cytoplasmic fraction in association with ribosomes, and fluorescence microscopy revealed a cytoplasmic localization of Trf4-1. Thus, oligoadenylation enhances exosomal mRNA degradation in the cytoplasm.

Identification of ALK gene alterations in urothelial carcinoma.

BACKGROUND: Anaplastic lymphoma kinase (ALK) genomic alterations have emerged as a potent predictor of benefit from treatment with ALK inhibitors in several cancers. Currently, there is no information about ALK gene alterations in urothelial carcinoma (UC) and its correlation with clinical or pathologic features and outcome. METHODS: Samples from patients with advanced UC and correlative clinical data were collected. Genomic imbalances were investigated by array comparative genomic hybridization (aCGH). ALK gene status was evaluated by fluorescence in situ hybridization (FISH). ALK expression was assessed by immunohistochemistry (IHC) and high-throughput mutation analysis with Oncomap 3 platform. Next generation sequencing was performed using Illumina Genome Analyzer IIx, and Illumina HiSeq 2000 in the FISH positive case. RESULTS: 70 of 96 patients had tissue available for all the tests performed. Arm level copy number gains at chromosome 2 were identified in 17 (24%) patients. Minor copy number alterations (CNAs) in the proximity of ALK locus were found in 3 patients by aCGH. By FISH analysis, one of these samples had a deletion of the 5'ALK. Whole genome next generation sequencing was inconclusive to confirm the deletion at the level of the ALK gene at the coverage level used. We did not observe an association between ALK CNA and overall survival, ECOG PS, or development of visceral disease. CONCLUSIONS: ALK genomic alterations are rare and probably without prognostic implications in UC. The potential for testing ALK inhibitors in UC merits further investigation but might be restricted to the identification of an enriched population.

Determination of sulfotransferase forms involved in the metabolic activation of the genotoxicant 1-hydroxymethylpyrene using bacterially expressed enzymes and genetically modified mouse models.

1-Methylpyrene, a carcinogenic polycyclic aromatic hydrocarbon, forms benzylic DNA adducts, in particular N2-(1-methylpyrenyl)-2'-deoxyguanosine, in mice and rats. It is bioactivated via 1-hydroxymethylpyrene (1-HMP) to electrophilic 1-sulfooxymethylpyrene (1-SMP). In this study, we explored the role of individual mouse sulfotransferase (SULT) forms in this activation. First, we showed that all nine mouse SULTs tested were able to activate 1-HMP to a mutagen in the his- Salmonella typhimurium reversion test. Some activation was even observed with Sult2a3 and Sult5a1, orphan forms for which no substrates were identified hitherto. Subsequently, we used cytosolic preparations from tissues of four mouse lines (wild-type, Sult1a1-, Sult1d1-, and transgenic for human SULT1A1/2) for the activation of 1-HMP in the mutagenicity assay. The most prominent impacts of the genetic SULT status were 96% decrease in hepatic activation by Sult1a1 knockout, 99% decrease in renal activation by Sult1d1 knockout, and 100-fold increase in pulmonary activation by transgenic human SULT1A1/2. Finally, we treated the various mouse lines with 1-HMP (19.3 mg/kg, intraperitoneally), and then determined 1-SMP levels in plasma and DNA adducts in tissues. Transgenic human SULT1A1/2 strongly enhanced 1-SMP plasma levels and DNA adduct formation in the liver, lung, heart, and kidney but not in the colon. Sult1a1 and Sult1d1 knockout reduced plasma 1-SMP levels as well as DNA adduct formation in some tissues (strongest effects: 97% decrease in 1-SMP and 89% decrease in hepatic adducts in Sult1a1- mice). The adduct levels detected in various tissues did not accurately reflect the activation capacity of these tissues determined in vitro, probably due to the distribution of the reactive metabolite 1-SMP via the circulation. In conclusion, we demonstrated that many mouse SULT forms are able to activate 1-HMP. In vivo, we verified a prominent role of Sult1a1 in hepatic and renal adduct formation and a smaller but unambiguous role of Sult1d1, and demonstrated the strong impact of transgenic human SULT1A1/2.

Formation of hepatic DNA adducts by methyleugenol in mouse models: drastic decrease by Sult1a1 knockout and strong increase by transgenic human SULT1A1/2.

Methyleugenol--a natural constituent of herbs and spices--is hepatocarcinogenic in rodent models. It can form DNA adducts after side-chain hydroxylation and sulfation. We previously demonstrated that human sulfotransferases (SULTs) 1A1 and 1A2 as well as mouse Sult1a1, expressed in Salmonella target strains, are able to activate 1'-hydroxymethyleugenol (1'-OH-ME) and 3'-hydroxymethylisoeugenol (3'-OH-MIE) to mutagens. Now we investigated the role of these enzymes in the formation of hepatic DNA adducts by methyleugenol in the mouse in vivo. We used FVB/N mice [wild-type (wt)] and genetically modified strains in this background: Sult1a1 knockout (ko), transgenic for human SULT1A1/2 (tg) and the combination of both modifications (ko-tg). Methyleugenol (50mg/kg body mass) formed 23, 735, 3770 and 4500 N (2)-(trans-methylisoeugenol-3'-yl)-2'-deoxyguanosine adducts per 10(8) 2'-deoxyribonucleosides (dN) in ko, wt, ko-tg and tg mice, respectively. The corresponding values for an equimolar dose of 1'-OH-ME were 12, 1490, 12 400 and 13 300 per 10(8) dN. Similar relative levels were observed for the minor adduct, N (6)-(trans-methylisoeugenol-3'-yl)-2'-deoxyadenosine. Thus, the adduct formation by both compounds was nearly completely dependent on the presence of SULT1A enzymes, with human SULT1A1/2 producing stronger effects than mouse Sult1a1. Moreover, a dose of 0.05 mg/kg methyleugenol (one-fourth of the estimated average daily exposure of humans) was sufficient to form detectable adducts in humanized (ko-tg) mice. Although 3'-OH-MIE was equally mutagenic to 1'-OH-ME in Salmonella strains expressing human SULT1A1 or 1A2, it only formed 0.14% of hepatic adducts in ko-tg mice compared with an equimolar dose of 1'-OH-ME, suggesting an important role of detoxifying pathways for this isomer in vivo.

Microarray and deep sequencing cross-platform analysis of the mirRNome and isomiR variation in response to epidermal growth factor.

BACKGROUND: Epidermal Growth Factor (EGF) plays an important function in the regulation of cell growth, proliferation, and differentiation by binding to its receptor (EGFR) and providing cancer cells with increased survival responsiveness. Signal transduction carried out by EGF has been extensively studied at both transcriptional and post-transcriptional levels. Little is known about the involvement of microRNAs (miRNAs) in the EGF signaling pathway. miRNAs have emerged as major players in the complex networks of gene regulation, and cancer miRNA expression studies have evidenced a direct involvement of miRNAs in cancer progression. RESULTS: In this study, we have used an integrative high content analysis approach to identify the specific miRNAs implicated in EGF signaling in HeLa cells as potential mediators of cancer mediated functions. We have used microarray and deep-sequencing technologies in order to obtain a global view of the EGF miRNA transcriptome with a robust experimental cross-validation. By applying a procedure based on Rankprod tests, we have delimited a solid set of EGF-regulated miRNAs. After validating regulated miRNAs by reverse transcription quantitative PCR, we have derived protein networks and biological functions from the predicted targets of the regulated miRNAs to gain insight into the potential role of miRNAs in EGF-treated cells. In addition, we have analyzed sequence heterogeneity due to editing relative to the reference sequence (isomiRs) among regulated miRNAs. CONCLUSIONS: We propose that the use of global genomic miRNA cross-validation derived from high throughput technologies can be used to generate more reliable datasets inferring more robust networks of co-regulated predicted miRNA target genes.

Highly diverse chromoviruses of Beta vulgaris are classified by chromodomains and chromosomal integration.

BACKGROUND: Chromoviruses are one of the three genera of Ty3-gypsy long terminal repeat (LTR) retrotransposons, and are present in high copy numbers in plant genomes. They are widely distributed within the plant kingdom, with representatives even in lower plants such as green and red algae. Their hallmark is the presence of a chromodomain at the C-terminus of the integrase. The chromodomain exhibits structural characteristics similar to proteins of the heterochromatin protein 1 (HP1) family, which mediate the binding of each chromovirus type to specific histone variants. A specific integration via the chromodomain has been shown for only a few chromoviruses. However, a detailed study of different chromoviral clades populating a single plant genome has not yet been carried out. RESULTS: We conducted a comprehensive survey of chromoviruses within the Beta vulgaris (sugar beet) genome, and found a highly diverse chromovirus population, with significant differences in element size, primarily caused by their flanking LTRs. In total, we identified and annotated full-length members of 16 families belonging to the four plant chromoviral clades: CRM, Tekay, Reina, and Galadriel. The families within each clade are structurally highly conserved; in particular, the position of the chromodomain coding region relative to the polypurine tract is clade-specific. Two distinct groups of chromodomains were identified. The group II chromodomain was present in three chromoviral clades, whereas families of the CRM clade contained a more divergent motif. Physical mapping using representatives of all four clades identified a clade-specific integration pattern. For some chromoviral families, we detected the presence of expressed sequence tags, indicating transcriptional activity. CONCLUSIONS: We present a detailed study of chromoviruses, belonging to the four major clades, which populate a single plant genome. Our results illustrate the diversity and family structure of B. vulgaris chromoviruses, and emphasize the role of chromodomains in the targeted integration of these viruses. We suggest that the diverse sets of plant chromoviruses with their different localization patterns might help to facilitate plant-genome organization in a structural and functional manner.

Identification of a mutation in the extracellular domain of the Epidermal Growth Factor Receptor conferring cetuximab resistance in colorectal cancer.

Antibodies against epidermal growth factor receptor (EGFR)--cetuximab and panitumumab--are widely used to treat colorectal cancer. Unfortunately, patients eventually develop resistance to these agents. We describe an acquired EGFR ectodomain mutation (S492R) that prevents cetuximab binding and confers resistance to cetuximab. Cells with this mutation, however, retain binding to and are growth inhibited by panitumumab. Two of ten subjects studied here with disease progression after cetuximab treatment acquired this mutation. A subject with cetuximab resistance harboring the S492R mutation responded to treatment with panitumumab.

Palaeohexaploid ancestry for Caryophyllales inferred from extensive gene-based physical and genetic mapping of the sugar beet genome (Beta vulgaris).

Sugar beet (Beta vulgaris) is an important crop plant that accounts for 30% of the world's sugar production annually. The genus Beta is a distant relative of currently sequenced taxa within the core eudicotyledons; the genomic characterization of sugar beet is essential to make its genome accessible to molecular dissection. Here, we present comprehensive genomic information in genetic and physical maps that cover all nine chromosomes. Based on this information we identified the proposed ancestral linkage groups of rosids and asterids within the sugar beet genome. We generated an extended genetic map that comprises 1127 single nucleotide polymorphism markers prepared from expressed sequence tags and bacterial artificial chromosome (BAC) end sequences. To construct a genome-wide physical map, we hybridized gene-derived oligomer probes against two BAC libraries with 9.5-fold cumulative coverage of the 758 Mbp genome. More than 2500 probes and clones were integrated both in genetic maps and the physical data. The final physical map encompasses 535 chromosomally anchored contigs that contains 8361 probes and 22 815 BAC clones. By using the gene order established with the physical map, we detected regions of synteny between sugar beet (order Caryophyllales) and rosid species that involves 1400-2700 genes in the sequenced genomes of Arabidopsis, poplar, grapevine, and cacao. The data suggest that Caryophyllales share the palaeohexaploid ancestor proposed for rosids and asterids. Taken together, we here provide extensive molecular resources for sugar beet and enable future high-resolution trait mapping, gene identification, and cross-referencing to regions sequenced in other plant species.

Exome sequencing identifies recurrent mutations of the splicing factor SF3B1 gene in chronic lymphocytic leukemia.

Here we perform whole-exome sequencing of samples from 105 individuals with chronic lymphocytic leukemia (CLL), the most frequent leukemia in adults in Western countries. We found 1,246 somatic mutations potentially affecting gene function and identified 78 genes with predicted functional alterations in more than one tumor sample. Among these genes, SF3B1, encoding a subunit of the spliceosomal U2 small nuclear ribonucleoprotein (snRNP), is somatically mutated in 9.7% of affected individuals. Further analysis in 279 individuals with CLL showed that SF3B1 mutations were associated with faster disease progression and poor overall survival. This work provides the first comprehensive catalog of somatic mutations in CLL with relevant clinical correlates and defines a large set of new genes that may drive the development of this common form of leukemia. The results reinforce the idea that targeting several well-known genetic pathways, including mRNA splicing, could be useful in the treatment of CLL and other malignancies.

Altered tissue distribution of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-DNA adducts in mice transgenic for human sulfotransferases 1A1 and 1A2.

Soluble sulfotransferases (SULTs) generate electrophilically reactive metabolites from numerous food-borne compounds, environmental contaminants and drugs, often resulting in mutagenicity and carcinogenicity. Substrate specificity, regulation and tissue distribution of SULTs show large interspecies differences. In humans, therefore, SULTs may be involved in the induction of cancer in different tissues than in standard animal models. To construct a rodent model taking some species differences into account, we transferred a 68.5 kb human (h) genomic sequence that comprised the transcribed and long flanking regions of SULT1A1 and 1A2 into murine oocytes. This approach resulted in several mouse lines expressing these human genes in a copy number-dependent manner with a tissue distribution similar to that in humans. In previous in vitro studies, we had demonstrated that human SULT1A1 and 1A2 efficiently catalyze the terminal activation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to a mutagen. The transgenic mice were used to study the hSULT1A1/1A2-mediated activation. Tissue distribution and levels of DNA adducts were determined in hSULT1A1/1A2 transgenic and wild-type mice after an oral dosage of PhIP. Transgenic mice exhibited significantly elevated PhIP-DNA adduct levels compared with the wild-type in liver (13-fold), lung (3.8-fold), colon (2-fold), kidney (1.6-fold) and cecum (1.5-fold). Moreover, among the eight tissues examined, liver was the one with the lowest and highest adduct levels in wild-type and transgenic mice, respectively. Hence, expression of hSULT1A1/1A2 not only enhanced the genotoxicity but also substantially changed the organotropism of PhIP.

Whole-genome sequencing identifies recurrent mutations in chronic lymphocytic leukaemia.

Chronic lymphocytic leukaemia (CLL), the most frequent leukaemia in adults in Western countries, is a heterogeneous disease with variable clinical presentation and evolution. Two major molecular subtypes can be distinguished, characterized respectively by a high or low number of somatic hypermutations in the variable region of immunoglobulin genes. The molecular changes leading to the pathogenesis of the disease are still poorly understood. Here we performed whole-genome sequencing of four cases of CLL and identified 46 somatic mutations that potentially affect gene function. Further analysis of these mutations in 363 patients with CLL identified four genes that are recurrently mutated: notch 1 (NOTCH1), exportin 1 (XPO1), myeloid differentiation primary response gene 88 (MYD88) and kelch-like 6 (KLHL6). Mutations in MYD88 and KLHL6 are predominant in cases of CLL with mutated immunoglobulin genes, whereas NOTCH1 and XPO1 mutations are mainly detected in patients with unmutated immunoglobulins. The patterns of somatic mutation, supported by functional and clinical analyses, strongly indicate that the recurrent NOTCH1, MYD88 and XPO1 mutations are oncogenic changes that contribute to the clinical evolution of the disease. To our knowledge, this is the first comprehensive analysis of CLL combining whole-genome sequencing with clinical characteristics and clinical outcomes. It highlights the usefulness of this approach for the identification of clinically relevant mutations in cancer.

p53 Gene repair with zinc finger nucleases optimised by yeast 1-hybrid and validated by Solexa sequencing.

The tumor suppressor gene p53 is mutated or deleted in over 50% of human tumors. As functional p53 plays a pivotal role in protecting against cancer development, several strategies for restoring wild-type (wt) p53 function have been investigated. In this study, we applied an approach using gene repair with zinc finger nucleases (ZFNs). We adapted a commercially-available yeast one-hybrid (Y1H) selection kit to allow rapid building and optimization of 4-finger constructs from randomized PCR libraries. We thus generated novel functional zinc finger nucleases against two DNA sites in the human p53 gene, near cancer mutation 'hotspots'. The ZFNs were first validated using in vitro cleavage assays and in vivo episomal gene repair assays in HEK293T cells. Subsequently, the ZFNs were used to restore wt-p53 status in the SF268 human cancer cell line, via ZFN-induced homologous recombination. The frequency of gene repair and mutation by non-homologous end-joining was then ascertained in several cancer cell lines, using a deep sequencing strategy. Our Y1H system facilitates the generation and optimisation of novel, sequence-specific four- to six-finger peptides, and the p53-specific ZFN described here can be used to mutate or repair p53 in genomic loci.

Multiple platform assessment of the EGF dependent transcriptome by microarray and deep tag sequencing analysis.

BACKGROUND: Epidermal Growth Factor (EGF) is a key regulatory growth factor activating many processes relevant to normal development and disease, affecting cell proliferation and survival. Here we use a combined approach to study the EGF dependent transcriptome of HeLa cells by using multiple long oligonucleotide based microarray platforms (from Agilent, Operon, and Illumina) in combination with digital gene expression profiling (DGE) with the Illumina Genome Analyzer. RESULTS: By applying a procedure for cross-platform data meta-analysis based on RankProd and GlobalAncova tests, we establish a well validated gene set with transcript levels altered after EGF treatment. We use this robust gene list to build higher order networks of gene interaction by interconnecting associated networks, supporting and extending the important role of the EGF signaling pathway in cancer. In addition, we find an entirely new set of genes previously unrelated to the currently accepted EGF associated cellular functions. CONCLUSIONS: We propose that the use of global genomic cross-validation derived from high content technologies (microarrays or deep sequencing) can be used to generate more reliable datasets. This approach should help to improve the confidence of downstream in silico functional inference analyses based on high content data.

Expression analysis of proline rich 15 (Prr15) in mouse and human gastrointestinal tumors.

Proline rich 15 (Prr15), which encodes a protein of unknown function, is expressed almost exclusively in postmitotic cells both during fetal development and in adult tissues, such as the intestinal epithelium and the testis. To determine if this specific expression is lost in intestinal neoplasias, we examined Prr15 expression by in situ hybridization (ISH) on mouse intestinal tumors caused by different gene mutations, and on human colorectal cancer (CRC) samples. Prr15/PRR15 expression was consistently observed in mouse gastrointestinal (GI) tumors caused by mutations in the Apc gene, as well as in several advanced stage human CRCs. In contrast, no Prr15 expression was detected in intestinal tumors derived from mice carrying mutations in the Smad3, Smad4, or Cdkn1b genes. These findings, combined with the fact that a majority of sporadic human CRCs carry APC mutations, strongly suggest that the expression of Prr15/PRR15 in mouse and human GI tumors is linked, directly or indirectly, to the absence of the APC protein or, more generally, to the disruption of the Wnt signaling pathway.

ojoplano-mediated basal constriction is essential for optic cup morphogenesis.

Although the vertebrate retina is a well-studied paradigm for organogenesis, the morphogenetic mechanisms that carve the architecture of the vertebrate optic cup remain largely unknown. Understanding how the hemispheric shape of an eye is formed requires addressing the fundamental problem of how individual cell behaviour is coordinated to direct epithelial morphogenesis. Here, we analyze the role of ojoplano (opo), an uncharacterized gene whose human ortholog is associated with orofacial clefting syndrome, in the morphogenesis of epithelial tissues. Most notably, when opo is mutated in medaka fish, optic cup folding is impaired. We characterize optic cup morphogenesis in vivo and determine at the cellular level how opo affects this process. opo encodes a developmentally regulated transmembrane protein that localizes to compartments of the secretory pathway and to basal end-feet of the neuroepithelial precursors. We show that Opo regulates the polarized localization of focal adhesion components to the basal cell surface. Furthermore, tissue-specific interference with integrin-adhesive function impairs optic cup folding, resembling the ocular phenotype observed in opo mutants. We propose a model of retinal morphogenesis whereby opo-mediated formation of focal contacts is required to transmit the mechanical tensions that drive the macroscopic folding of the vertebrate optic cup.

Mouse splice mutant generation from ENU-treated ES cells--a gene-driven approach.

Mutant mice are important for elucidating mammalian gene functions and for modeling human disease phenotypes. In recent years, chemical mutagenesis has become an increasingly popular method to disrupt gene functions due to its high efficiency of inducing mutations throughout the genome. Mutagenesis of embryonic stem (ES) cells offers the possibility of gene-driven approaches, which, however, require efficient mutation detection procedures to screen archives of mutated samples for lesions in particular genes. We have developed an approach that focuses on the detection of splice mutations in highly pooled cDNA samples using exon-skipping PCR primers. As a proof of concept, splice mutants for the Kit gene were isolated from a library comprising approximately 40,000 ES cell clones treated with N-ethyl-N-nitrosourea followed by transmission through the mouse germ-line. The approach will be useful for the production of mouse models for human disease-related splice mutations and as a general gene disruption strategy.